The RNeasy UCP Micro Kit is designed for purification of up to 45 μg RNA from small or low biomass samples. The spin columns and buffers are treated to remove exogenous nucleic acids.
RNeasy UCP Micro technology combines the selective binding properties of an ultra-clean, silica-based membrane with the speed of microspin technology. Ultra-clean guanidine-thiocyanate–containing lysis buffer RULT and ethanol are added to the sample to create conditions that promote selective binding of RNA to the RNeasy UCP MinElute membrane. The sample is then applied to the RNeasy UCP MinElute spin column and RNA binds to the silica membrane. Traces of DNA that may copurify are removed by DNase treatment on the RNeasy UCP MinElute spin column. DNase and any contaminants are washed away with ultra-clean wash buffers RUWT and RUPE, and high-quality total RNA is eluted in ultra-clean water (see figure RNeasy UCP Micro Kit Procedure). With the RNeasy UCP Micro procedure, all RNA molecules longer than 200 nucleotides are purified. The procedure enriches for mRNA, since most RNAs <200 nucleotides are selectively excluded.
Tissue samples can be conveniently stabilized using RNAlater RNA Stabilization Reagent or Allprotect Tissue Reagent, and efficiently disrupted using a TissueRuptor or TissueLyser system.

Total RNA was purified from 10‐fold serial dilutions of Jurkat cells
using the RNeasy UCP Micro Kit. The β‐actin transcript was detected even down to one cell by real‐time
RT‐PCR, and reproducible CT values were observed at each dilution, indicating the absence of inhibition.

Minimized residual nucleic acids.

Sample preparation was performed with mock samples to check for possible nucleic acid contamination in the kit. Three independent experiments with mock preparations were done with 3 different lot numbers for the RNeasy Micro Kit, as well
as for the RNeasy UCP Micro Kit. Eluates were analyzed by RT-PCR using primers for a
conserved bacterial 16S rRNA sequence. Delta CT values between the no template contro (NTC) and the empty
preparation were calculated for both kits. The delta CT value for the RNeasy UCP Micro Kit was lower compared to the delta CT of the RNeasy Micro Kit, showing that there are
minimal contaminants in the RNeasy UCP Micro Kit.

RNeasy UCP Micro Kit procedure.

β-actin PCR with and without the RT step to detect DNA.

Total RNA was purified from 10-fold serial dilutions of Jurkat cells (5 x 105 – 5 cells) using the RNeasy UCP Micro Kit. The β-actin transcript was detected inas few as 5 cells by real-time RT-PCR. In control reactions without reverse transcriptase (-RT), β-actin DNA
was not detected after 40 cycles, indicating the absence of genomic DNA
contamination.

Performance

The RNeasy UCP Micro Kit delivers highly reproducible yields of RNA (>200 nt) from small samples. RNA is reliably purified from small numbers of cells, as well as from small amounts of standard tissues. The RNeasy UCP Micro Kit provides maximum sensitivity in quantitative gene expression analyses, such as real-time RT-PCR and NGS, by efficient on-column digestion of genomic DNA. Ultra-clean production minimizes the risk of copurification and subsequent analysis of exogenous nucleic acids.

Principle

The RNeasy UCP Micro Kit is designed for isolation of total RNA from small cell or tissue samples and low biomass samples, and for use in sensitive applications such as NGS deep sequencing.
The kit components are decontaminated to minimize noise from exogenous DNA / RNA and increase reads of sample DNA / RNA in NGS.
RNeasy technology simplifies total RNA isolation by combining the stringency of guanidine-isothiocyanate lysis with the speed and purity of silica-membrane purification. The unique design of the RNeasy MinElute spin columns enables minimal elution volumes, as little as 10 µl, to concentrate the RNA. The small elution volume means that reaction volumes can be kept to a minimum in downstream applications, increasing reaction efficiency. High purity with minimal residual background from exogenous nucleic acids allows the complete sample to be used in downstream reactions such as NGS.

Procedure

The RNeasy UCP Micro Kit is designed for isolating total RNA from small samples. Samples (<5 mg tissue, or <5 x 105 cells) are first lysed and then homogenized. Ethanol is added to the lysate to provide ideal binding conditions. The lysate is then loaded onto the RNeasy UCP MinElute spin column and RNA (up to 45 µg) binds to the silica membrane. RNase-free DNase, provided in the RNeasy UCP Micro Kit, enables simple and efficient on-column digestion of genomic DNA for sensitive applications. DNase and any contaminants are efficiently washed away and pure, concentrated RNA is eluted in 10–14 µl water.

Applications

Highly reproducible isolation of pure RNA in a small elution volume (10 µl) makes this kit suitable for sensitive, quantitative gene expression analyses, including real-time RT-PCR starting with as little as one cell.

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